OA12339A - Use of cox-2 inhibitors for preventing immunodeficiency. - Google Patents

Abstract

The present invention provides a method for treating or preventing a disorder typified by an immunodificiency (e.g. HIV), wherein the patient is administered a COX-2 inhibitor or derivative or pharmaceutically acceptable salt thereof, preferably diisopropylfluorophasphaate, L-745337, rofecoxi, NS 398, SC 58125, etodolac, meloxicam, celecoxib flusolide or nimesulide, and compositions and products containing the same or use of the same in preparing medicaments and for treatment.

Description

4- 012339

METHOD

The invention is in the field of treatment of5 immunodeficiencies and viral infections. More

specifically, the invention relates to the use ofcyclooxygenase-2 (COX-2) inhibitors. or dérivativesthereof in immunomodulation for treatment ofimmunodeficiency and viral diseases; especially HIV 10 infection and AIDS and related conditions.

Prostaglandine play an important rôle in theinflammation process and inhibition of^formation ofprostaglandine has been a popular target for developmentof anti-inflammatory drugs... Non-steroid anti- 15 inflammatory drugs (NSAID's) inhibit cyclooxygenase (COX) which is an enzyme involved in the biosynthesis ofprostaglandin interraediates from arachidonic acid.

There are several NSAID's in clinical use includingdrugs like indomethacin, piroxicam, tenoxicam, 20 diclofenac, meloxicam, tenidap, isoxicam, acetylsalicylic acid, diflunisal, sulindac, ibuprofen,naproxen and ketoprofen. NSAID's are today among- the most widely prescribed .drugs worldwide. 25 These NSAID's are clinically efficient drugs and they possess antipyretic, anti-inflammatory andantithrombotic effects. The main indications for thisclass of drugs are arthritis including osteoarthritisand rheumatoid arthritis, païnful musculoskeletal 30 conditions and general pain conditions. However, thereare severe side-effects with these drugs. The mostfrequent side effects are gastrointestinal ulcérationand bleeding, inhibition of platelet aggregation andinteraction with other drugs.

In the early 1990's a second COX isoform of theenzyme was cloned. This new COX isoform is now known asCOX-2 (Vane et al, 1998, Ann. Rev. Pharmacol. Toxicol., 35

I h 012339 38, p97-120).

There are now two well known isoforms of COX, COX-1and COX-2 (recently the existence of COX-3 has a^so beenpostulated). COX-1 ia présent in most tissues and canbe regarded as the housekeeper enzyme. The activity ofthe COX-l enzyme protects, for example, the lining inthe gastrointestinal tract. COX-2, however, is notprésent normally but increases during inflammation.Several of the aide effects of NSAID's are related toinhibition of COX-1 enzyme. NSAID’s inhibit both COX-1 —and COX-2 {see* Tables 1-3) :

Z

Table 1: IC50 values and COX-2/COX-1 ratios of differentNSAID's in guinea pig macrophage model (IC50 values fromEngelhart et al. in J. Inflammatory Res., 44, p422-43,1995) NSAID'S COX-2 ICSO (gmol/litre) COX-1 IC50 (junolZlitre) COX-2 selectivity COX-l/COX-2 Meloxicam 0.0019 0.00577 3 Diclofenac 0.0019 0.000855 0.45 Piroxicam 0.175 0.00527 0.030 Tcnoxicam 0.322 0.201 0.6 Indomethacin 0.00636 0.00021 0.03 Teridep 47.8 0.393 0.008 012339 - 3 -

Table 2 : IC50 values for NSAID's in intact cell on COX-1(bovine endothélial cells) and COX-2 (stimulatedmacrophages) (IC50 values from Taketo in J. NationalCancer Institute, 90, pl529-1536, 1998) NSAID'S COX-2 IC50(pmol/litre) COX-1 IC50(μιηοΐ/litre) COX-2 selectivity COX-l/COX-2 Asprin 50 0.3 0.006 Indomethacin 0.6 0.01 0.02 Tolfenamic acid 0.005 0.0003 0.06 Ibuprofen 15 1 0.07 Acetaminophen 20 2.7 0.¼ Sodium salicylate 100 35 0.35 BW 755C 1.2 0.65 0.5 Flubiprofen 0.025 0.02 0.8 Carprofen 3 3 1 Diclofenac 0.35 0.5 1.4 Naproxen 1.3 2.2 1.7 BF 389 0.03 0.15 5 012339'

IahiS-3-î, Inhibition of recombinant human PGH synthesis (COX-1 and COX-2) (IC50 values from Laneuvill et al. in J. Pharm. Exp. Ther., 271, p927-34, 1994) NSAID'S COX-2 IC50 (μτηοΐ/litre) COX-1 IC50 (funoWitre) COX-2 selectivity COX-l/COX-2 Indomcthacin >1000 13.5 <0.01 Sulindac sulphide 50.7 1.3 0.03 Piroxicam >500 17.7 0.04 Diclofenac «MF / 20.5 2.7 0.13 Hubiprofen 3.2 0.5 0.16 Mcclofcnemate 9.7 1.5 0.15 Phenylbutazone >100 16.0 <0.16 Naproxen 28.4 4.8 0.17 Ibuprofen 12.5 4.0 0.3 Ketorolac ttomethamine 60.5 31.5 0.5 DHA (22:6) 41 25.6 0.6 6-MNA 93.5 64,0 0.7 Etodolac 60 74.4 1.2 Salicyclic acid >1000 >1000 ~1

During the last decade several new sélective COX-2inhibitors and so called "preferential" COX-2 inhibitorshâve been identified. Several of these COX-2 inhibitorshâve been developed and a few of these hâve recentlyreached the market. Some of these new COX-2 inhibitorsdo not show inhibition of COX-1 in clinical doses.Extensive clinical studies and clinical practise on useof these COX-2 inhibitors show that these new COX-2inhibitors hâve great advantages with regard to safetycompared to non-selective NSAID’s. For reviews on COX-2inhibitors see for example Golden et al., 1999,

Osteoarthritis. 25, p359-379, Mitchel et al., 1999,

Brit. J. Pharmacol., 128, pll21-1132, Lipsky, 1999, Am. J. Med., 106 (5B), p515-575, Taketo, 1998, J. NationalCancer Inst., 90, pl529-1537, Griswold et al., 1996,

Med. Res. Rev., 16, pl81-206 and Reitz et al., 1995, 012339 - 5 -

Ann. Rep. Med. Chem., 30, pl79-188.

Further publications of interest on different COX-2inhibitors include for example: Lane, 1997, J.Rheumatol., 24 (suppl. 49), p20-24, Mehlish et al., 1998, Clin. Pharmacol. Ther., 63, pl-8, Zhao et al.,1997, Arthritis Rheum., 40 (suppl.), S88, Ehrich et al.,1997, Arthritis Rheum., 40 (suppl.), S93, Maziasz etal., 1997, Arthritis Rheum., 40 (suppl.), S195, Mengle-Gaw et al., 1997, Arthritis Rheum., 40 (suppl.), S93,Morrison, 1999, Clin. Ther., 21, p943-953, Chan et al., 1995, J. Pharmacol. Exp. Ther., 274, pl531-37-; Riendeauet al., 1997, Br. J. Pharmacol., 121, plÛ5-il7„ Black etal., 1999, J. Med. Chem., 42, pl274-81, Cuo et al., 1996, J. Biol. Chem., 271, pl9134-39, Geiss, 1999,

Scand. J. Rheumatol., 109 (suppl.), p31-37, Warner etal., 1999, PNAS USA, 96, p7563-68, Bjarnson et al., 1997, Scand. J. Gastroenterol., 32, pl26-130, Danneberget al., 1999, Semin. Oncol., 26, p499-504, Mitchell etal., 1993, PNAS USA, 90, pll693-97, Futaki et al., 1994,Prostaglandine, 47, p55-9, Futaki et al., 1993, J.

Pharm. Pharmacol., 45, p753-5, Masferrer et al., 1994,PNAS USA, 91, p3228-32, Klein et al., 1994, Biochem.Pharmacol., 48, pl605-10, Reitz et aï., 1994, J. Med.Chem., 37, p3878-81,* Seibert et al., '1994, PNAS USA, 91,P12013-17, Klein et al., 1996, Biochem. Pharmacol., 51,P285-90, Nantal et al., 1998, 9th Intern. ConférenceInflamm. Res. Assoc., Nov 1-5, Pennig et al., 1997, J.Med. Chem., 40, pl347-65 and Puig et al., 2000, J. Med.Chem., 43, p214-223.

COX-2 inhibitors are a relatively diverse group ofcorapounds from a Chemical structure point of view.Compounds which selectively inhibit COX-2 are describedin many patent documents of the last decade. Some ofthese are WO 94/26781, WO 94/20480, WO 94/13635, WO

95/00501, WO 94/27980, WO 94/15932, WO 95/21817, WO

95/15316, WO 96/06840, WO 96/03388, WO 96/03387, WO

96/03392, WO 96/25405, WO 96/24584, WO 96/03385, WO 012339 - 6 - 96/16934, WO 98/41516, WO 98/43966, WO 99/12930, EPO 673366, WO 98/41511, WO 98/47871, WO 99/20110, WO 99/23087,WO 99/14194, WO 99/14195, WO 99/15513 and WO 99/15503and in US patents numbers 5,380,738, 5,344,991,5,393,790, 5,434,178, 5,474,995, 5,475,018 and5,510,368.

Two compounds are currently launched, rofecoxib (4-(4-methylsulfonyl)phenyl) -3-phenyl-2 (5H) -furanone) (I)in Vioxx® and celecoxib (4-{5-(4-methylphenyl)-3-(trifluoromethyl) -lH-pyrazol-l-yl) -benzenesulfonamide).(II) in jCelebra® :

Rofecoxib is described in WO 93/0500501 from MerckFrosst Canada and further in Morrison, 1999, Clin.Ther., 21, p943-953, Chan et al., 1995, J. Pharmacol.Exp. Ther., 274, pl531-37 and in Nantel et al., 1998,supra.

Celecoxib is described by Geiss, 1999, Scand. J.Rheumatol., 109 (suppl.), p31-37 and by Penning et al.,1997, J. Med. Chem., 40, pl347-65. Celecoxib isdescribed to be 375-fold more sélective for COX-2compared to COX-1.

Several other COX-2 inhibitors hâve been evaluatedin biological Systems and some of these are BF 3 89 (III) , CGP 28232 (IV) , DFP, DFU (V) , DuP 697 (VI) ,etodolac (VII), FK 3311 (VIII), flosulide (IX), L- h 012339 745,337 (X), meloxicam (Mobic®, US 4233299, 4-hydroxy-2-methyl-N- (5-methyl-2-thiazolyl)-1,1 -dioxide-2H-1,2-benzothiazine-3-carboxamide) (XI), MF tricyclic (XII) ,nimesulide (XIII), NS-398 (XIV) and SC-58125 (XV) :

(V)

(VI) 012339

(XIII)

(XIV) 012339

Purther compounds described for COX-2 inhibition-'include S-2474 (from Shionogi, EP 595546, 5(E)-(3,5-di-,tert-butyl-4-hydroxy) benzylidene-2-ethyl-l, 2- isothiazolidine-1,1-dioxide) (XVI), JTE-522 or RWK-57504(4- (4-cyclohexyl-2-methyl-5-oxazolyl) -2-fluoro-benzenesulfonamide) (XVII), Darbufelone mesylate(Pfizer, WO 94/03448, monomethanesulfonate sait of 2-amino-5-((3,5-bis (1,l-dimethylethyl)-4-hydroxyphenyl)methylene-4 (5H)-thiazolone) (XVIII), 6089(from Kotobuki Pharmaceutical) (XIX) , Valdecoxib(Pharmacia, 4- (5-methyl-3-phenyl-4-isoxazolyl)-benzenesulfonamide) (XX), Paracoxib sodium (Pharmacia,sodium sait of N-( (4-(5-methyl-3-phenyl-4-isozazolyl)-phenyl)sulfonyl)-propanamide) (XXI), 4-(2-oxo-3-phenyl-2,3-dihydrooxazol-4-yl) -benzenesulfonamide (Almirall-Prodespharma) (XXII) and Etoricoxib (MK-633, Merck andCo.) :

(XVII) 012339 - 10 -

(XXII)

(XXD 012339 - 11 -

The above deseribed compounds form preferred COX-2inhibitors for use in the methods deseribed hereinafter.

The indications for COX-2 inhibitors are arthritis,musculoskeletal pain conditions and general pain which 5 hâve been treated with classical NSAIDs such as indomethacin, diclofenac and naproxen. Recently, it hasalso been suggested to use COX-2 inhibitors in cancertherapy and maybe also cancer prévention. COX-2inhibitors might also hâve potential for use in relation 10 to Alzheimer disease and other dementia-associated brain processes. ~~

The potentials of the clinical utility of COX-2 £. inhibitors are discussed in for example Nature, 367,p215-216 (1994), in Drug News and Perspectives, 7, p501- 15 512 (1994), in Annual Reports in Médicinal Chem., 30, pl79-188 (1995) and in Oncogene, 18, p7908-7916 (1999).

There are no spécifie suggestions for use of COX-2inhibitors in antiviral therapy or more specifically inHIV/AIDS therapy, and no COX-2 inhibitors hâve been 20 tested with regard to. anti-HIV effects. Furthermore,there is no suggestion to use COX-2 inhibitors (ornon-sélective COX-inhibitors) as immunostimulatoryagents in the treatment of immunodeficiency of viral andnon-viral origin. 25 HIV infection and AIDS is a major health problem with more than 33 million people infected with the virusworldwide. Most of the infected people are located inAfrica (sub-Sahara) and in parts of Asia. There aretoday two classes of anti-AIDS compounds in routine 30 clinical use; inhibitors of HIV reverse transcriptaseand inhibitors of HIV protease. HIV reversetranscriptase inhibitors can be divided into non-nucleoside reverse transcriptase inhibitors (NNRTIs) andnucleoside reverse transcriptase inhibitors (NRTIs).

The most frequently used NNRTI's are nevirapine,delavirdine, efavirenz, emivirine and T180. The mostfrequently used NRTI's include zidovudine, didanosine, 35 012339 - 12 - stavudine and zalcitabine. Clinically useful HIVprotease inhibitors include inclinavir, palinavir andsaquiravir.

The présent treatment of HIV infection and AIDS is5 based on a combination of several drugs, a so-called cocktail of inhibitors of reverse transcriptase andprotease inhibitors. These combinations, called HAART(highly active antirétroviral therapy), are quiteeffective and can reduce the virus back to undetectable 10 levels in patient*s blood. However, HAART is not a curefor the patient, because the virus is still présent inthe immune cells, and tjie disease can reappear at anytime; upon discontinuation of therapy viremia peaks andrapid progression to AIDS is frequently observed. 15 Furthermore, the immunodeficiency and the HIV-specificT-cell dysfunction persiste during HAART. This therapyrequires life-long treatment and the treatment is veryexpensive, . The cost of the drugs alone, often exceedsUSD 15 000. There are, in addition, several other 20 problems associated with this therapy; difficulties withpatient compliance (complicated drug régimes),'development of résistant viruses, non-idealpharmacokinetics and side effects such as, for example,suppression of bone-marrow and long-term metabolic 25 effects.

For recently published reviews on anti-HlV therapysee for example: Hilgegroth, 1998, Pharm. uns. Zeit.,1998, 27, p22-25, Hilgegroth, 1998, Pharm. uns Zeit., 7,plll-116, Stellbrink, 1997, Dk Ârztebl., 94, p2497-2503, 30 Rettle et al., 1998, Int. J. STD AIDS, 9, p80-87, De-

Clercq, 1998, Antiviral Res., 38, pl53-179, Gait et al.,1995, TIBTECH, 13, p430-438 and RedBhaw et al. in"Emerging Drugs: The Prospects of Improved Medicines",Chapter 6, pl27-154, 1997. 35 In conclusion, although multidrug combinations like HAART has significantly improved the prognosis for patients suffering from HIV infection, there is a 1 h 012339 - 13 - medical need for new compounds in antiviral therapy ofHIV; especially agents stimulating the immune eystem.

The présent invention addresses this need.

Expression of COX-2 is normally restricted to 5 brain/brain processes, to arthritic synovia and sites oftissue injury. COX-2 is not found in normal lymph nodesor lymphocytes. It has now surprisingly been foundhowever that in mice infected by thé immunodeficiencydisorder MAIDs, lymph node cells express high levels of

10 COX-2. Furthermore, positively selected CD4+ and CD8+ T cells as well ,èts B cells from MAIDS lymph nodes ~~ contained high levels of COX-2 (see Example 2). It has l. been found that this COX-2 may be targetted to alleviatesymptoms of the immunodeficiency disorder, e.g. to 15 alleviate T cell dysfunction by acting as an immunostimulant, e.g. by generating antigen-spécifieimmune responses.

Whilst not wishing to be bound by theory, it isbelieved that COX-2 activity increases PGE2 production 20 which in turn increases the levels of cAMP whichactivâtes the PKA signalling pathway resulting inimpaired lymphocyte function. Work conducted on micewith MAIDs in vivo illustrâtes that COX-2 inhibitorsimprove the immune functions of T cells (see Example 6) . 25 The présent invention provides a new method for treating or preventing immunodeficiency; especially fortreatment of HIV and AIDS which comprises treating asubject with a therapeutically effective amount of aCOX-2 inhibitor or dérivative or pharmaceutically 30 acceptable sait thereof.

Thus in a first aspect the présent invention provides a method of treating or preventing a disordertypified by increased COX-2 activity, such as disorderstypified by decreased immune function, in a human or 35 non-human animal (e.g. through increased COX-2 expression) wherein said animal is administered atherapeutically effective amount of a COX-2 inhibitor or t 012339 - 14 - dérivative or pharmaceutically acceptable sait thereof.As used herein increased COX-2 activity refers to increased levels of activity either through theproduction of more COX-2 molécules (e.g. increased 5 expression), and/or more active molécules (e.g. conversion from latent to active forms or removal ofinhibition of the active -form) . Preferably saiddisorder is typified by decreased immune function, ie.is a condition of immunodeficiency e.g. exhibits 10 lymphocyte dysfunctions. As used herein "immunodeficiency" refers~to impaired function of cellsinvolved in normal immune rssponses, particularly B andT cells. Thus compounds described herein may be used toachieve immunostimulatory effects to enhance immune 15 responses. Thus COX-2 inhibitors are considered to hâveimmunomodulatory effects. Preferably conditions whichmay be treated include viral ly-induced immunodef iciencydisorders.

Thus, the method above would be useful for, but not 20 limited to, the treatment of HIV or AIDS related disorders in a subject. For example, approximately 50%of patients with common variable immunodef iciency hâve aT-cell dysfunction similar to that of HIV infection andcould benefit from immunostimulatory treatment. 25 According to the présent invention, any COX-2 inhibitormay be administered to a subject in need of HIV/AIDStherapy. Thus preferred conditions for treatmentaccording to the invention include infection byretroviruses, particularly HIV (and infection by related 30 viruses in other animais, e.g. SIV, FIV, MAIDS) and therésultant AIDS and treatment of common variableimmunodeficiency and related conditions to theaforementioned conditions.

Subjects which may be treated are preferably 35 mammalian, preferably humans and companion or agricultural animais such as dogs, cats, monkeys,horses, sheep, goats, cows, rabbits, rats and mice.

I 012339 - 15 -

Alternatively stated, the présent inventionprovides a COX-2 inhibitor or dérivative orpharmaceutically acceptable sait thereof for treating orpreventing a disorder typified by increased COX-2activity as described above or the use of a COX-2inhibitor or dérivative or pharmaceutically acceptablesait thereof in the préparation of a médicament fortreating or preventing a disorder typified by increasedCOX-2 activity as described above. As used herein"treating” refers to the réduction or alleviation,preferably to hormal levels, of one or more of thesymptoms of said disorder, e.g. infectivity or aréduction or alleviation of immune dysfunction."Preventing" refers to absolute prévention, i.e. absenceof détectable infectious agent, e.g virus and/ormaintenance of normal levels with reference to aparticular symptom (e.g. COX-2 activity) or réduction oralleviation of the extent or timing (e.g. delaying) ofthe onset of that symptom.

The enzyme cyclooxygenase 2 is a new target forHIV/AIDS therapy. The term "COX-2 inhibitor" dénotés acompound able to inhibit the enzyme cyclooxygenase 2without significant inhibition of cyclooxygenase 1 whenadministered at a particular concentration. Preferably,it includes compounds having a selectivity forcyclooxygenase-2 inhibition relative to cyclooxygenase-1inhibition (e.g. as determined by the COX-1:COX-2 IC80ratio according to the WHMA test, see below) of at least10, more preferably of at least 50, and even morepreferably of at least 100. (The selectivity ratio forone spécifie compound will vary with the biologicalassay and the form in which it is expressed (preferablyexpressed as the ratio of COX-1: COX-2 IC50 or IC80) , seetables 1-4) . The ratios described here refer to dataobtained in one or more relevant, well known COX assays,preferably using purified human enzymes, e.g. ratio ofIC50 values for example as determined by Engelhart et 012339 - 16 - al., 1995, supra. Preferably however, the test is theWHMA test as described below. A number of analyses of relative potencies of COX-1and CÔX-2 hâve been performed using a wide range ofassay Systems from isolated purified enzymes to intactcelle and cell models from various species. However, atprésent, the most widely accepted model is the humanwhole blood assay (WBA) and a modified version WilliamHarvey human modified whole blood assay (WHMA) which isthe preferred assay. These assays make use of readilyavailable humaji cells for testing which is préférablefor human use of the compounds. ^It also takes intoaccount the binding of NSAIDs to plasma proteins.Furthermore, assessment of selectivity is preferablymade at IC80 rather than at IC50 as the concentrationcurves for inhibition of COX-2 and COX-1 are notparallel and since most compounds are used at dosesgiving steady-state plasma concentrations of doser to80% inhibition (Warner et,al., 1999, PNAS USA, 96,p7563-7568)

In the WBA assay, for COX-1 analysis blood istreated with test agent followed 60 min later by calciumionophore and incubated for 3 0 min after which plasma iscollected. For COX-2 analysis, blood is treated withaspirin to inhibit COX-1 and 6 hours later with lipopolysaccharide and test agent and incubated for 18hours after which plasma is collected. Subséquently,the content of thromboxane B2 in plasma is assessed byradioimmunoassay as a measure of COX activity.

In the WHMA assay, COX-1 analysis is conducted asabove. For COX-2 analysis, blood is treated withconditioned medium from cultures of human airwayepithelium cells (A549) exposed to interleukin ΐβ for 24hours and incubated with this medium together with testagent for 60 min after which calcium ionophore is addedfollowed 30 min later by diclofenac to stop productionof prosanoids. Subsequently, plasma is collected and 012339 - 17 - analysed for its content of prostaglandin E2 in plasmaby radioimmunoassay as a measure of COX-2 activity. Thetimes of incubation for assessment of COX-l and COX-2activities are similar in this last assay which makes 5 activities more comparable and the WHMA the preferredassay.

Using this assay, selectivity based onCOX-2/WHMA-COX-l at IC80 is shown in Table 4 where 0.2and 0.02 represents 5- and 50-fold selectivities for 10 COX-2, respectively. 15 20

Table 4 : (Ratio COX-2 ! COX-l at IC8o according to the WHMAtest taken from Warner, et al., supra) :

Compouud Ratio COX-2/WHMA-COX-1at ICgQ Diisopropylfluorophosphate <0.01 L-745337 <0.01 rofecoxib 0.015 NS398 <0.05 SC58125 <0.01 (WBA assay) etodolac 0.043 meloxicam 0.091 celecoxib 0.11 nimesulide 0.17 25 30

In a preferred feature therefore the selectivityratio is determined according to the WHMA assay at IC80and compounds having a selectivity ratio of COX-2.-COX-1 of less than 0.2, preferably less than 0.05, e.g.less than 0.02, preferably less than 0.01, e.g. <0.005are particularly preferred for use in methods of theinvention. Alternatively stated, preferred compoundshâve a COX-l:COX-2 selectivity ratio (according to theWHMA assay at ICS0) of more than 2, preferably more than5, especially preferably more than 50 or 100, as 35 012339 - 18 - mentioned previously. "Inhibition" as referred to herein refers to aréduction in measurable cyclooxygenase-2 activity. Thismay be achieved by affecting transcription, translation,post-translational modification or activity of COX-2.Preferably however inhibition is achieved by inhibitingthe enzymatic activity, i.e. interfering with the activesite of pre-existing active COX-2 molécules.

Preferably, COX-2 inhibitors for treatment ofimmunodeficiency or viral infection, especially HIVinfections and,'AIDS, hâve a COX-2 -ÏCSO of less than about0.5 μπιοΐ/litre, more preferably less, than about 0.2μιηοΐ/litre.

The method provided herein relates to the use ofCOX-2 inhibitors or dérivatives thereof in theprévention and treatment of various conditions,including immuno-deficiencies and viral infections;especially HIV and AIDS.

In one preferred embodiment of the présentinvention, the COX-2 inhibitor for treatment accordingto the invention is selected from acidic sulfonamides.

In one preferred embodiment, COX-2 inhibitors foruse in the invention are selected from the compoundsaccording to the general formula A below includingmethansulphonamide ethers and thioethers: NHSO2CH3

R4 wherein X represents an oxygen or sulphur atom or alkyl group,preferably a -CH2- group; 012339 - 19 -

Rx represents a cycloalkyl or aryl group which mayoptionally be substituted by one or more groupa oratoms, preferably by one or more halogen atoms, such asfluorine; 5 R2, R3, R„ and Rs independently represent a hydrogen atom,a nitro or acyl group or an alkyl group which mayoptionally be substituted by one or more groups (e.g. anacyl group) or atoms or alternatively Rz and R3, R3 and R4 10 or R4 and Rs together with the intervening carbon atoms— form a cyclopentanone group; or a dérivative or a pharmaceutically acceptable saitthereof. 15

Preferably in such compounds X is an oxygen atom.

In further preferred compounds Rx is an aryl group or anaryl group substituted with one or more fluorine atoms,or a cycloalkyl group. 20 In further preferred compounds Ra and R3 are hydrogen atoms and R4 is an -NO2 or -COCH3 group.Alternative preferred compounds comprise those in whichR2 is a hydrogen atom and R3 and R4 together form acyclopentanone group. 25 Especially preferably compounds of formula A for use in the invention are compounds described hereindenoted flosulide, NS-398, nimesulide, FK 3311 and L-745337.

In another preferred embodiment of the présent 30 invention, the COX-2 inhibitor for use in the inventionis selected frora diaryl heterocycles.

One example of a family of diaryl· heterocycleswhich may be used as COX-2 inhibitors for use in theinvention comprises compounds of the general formula B 35 below 012339

wherein Y represents a cyclic group, preferably selected fromoxazolyl, isoxàzolyl, thienyl, dihydroéuryl, furyl,pyrrolyl, pyrazolyl, thiazolyl, imidazolyl,isothiazolyl, cyclopentenyl, phenyl and pyridyl; n is an integer from 0 to 3; m is an integer from 0 to 4; R6 represents a ketocyclyl, cycloalkyl or aryl group,which group may optionally be subs ti tut ed by one or moregroupe or atoms, preferably by one or more halogenatoms, such as fluorine; R·, each independently represent a substituent which maybe any functional group, preferably a hydrogen orhalogen atom, preferably fluorine or bromine, or analkyl group (preferably -CH3) , which alkyl group may besubstituted by one or more groupe or atoms, preferablyone or more fluorine atoms for'example -CF3; R8 represents an alkyl group, preferably -CH3 or NHRi0,preferably -NH2;

Rg represents a halogen atôm, preferably fluorine; and R10 represents a hydrogen atom or an alkyl group optionally substituted by one or more groups or atoms, 012339 - 21 - preferably by an acyl group; or a dérivative or a pharmaceutically acceptable saitthereof.

This class of compounds is claimed as anti-angiogenic agents in US 6,025,353 and a furtherdescription of preferred substituents and compoundsaccording to the présent invention are the same as in US6,025,353. — Preferabl,ÿ in such compounds Re is -NH2 or -CH3. Infurther preferred compounds Y is a pyrazolyl, furyl orthienyl group. Preferably R6 is an aryl group optionallysubstituted with one or more fluorine atoms. Preferablyn is 1 or 2. Preferably R7 is a bromine atom, an acylgroup or a substituted alkyl group such as-CF3.

Especially preferred compounds of formula B for usein the invention are compounds described herein denotedcelecoxib, rofecoxib, DuP-697, SC-58125, DFP, DFU, CGP28232 and MF tricyclic.

As used herein, the term "alkyl" includes any longor short chain, straight-chained, branched or cyclicaliphatic saturated or unsaturated hydrocarbon groupoptionally mono or poly substituted by hydroxy, alkoxy,acyloxy, nitro, alkoxycarbonyloxy, amino, aryl, oxo orhalo groups unless specifically stated otherwise. Theunsaturated alkyl groups may be mono- or polyunsaturatedand include both alkenyl and alkynyl groups. Suchgroups may contain up to 40, but preferably 1 to 10carbon atoms.

As used herein cyclic rings are preferably C5.7 andoptionally contain one or more heteroatoms selected fromoxygen, nitrogen and sulphur.

The term "acyl" as used herein includes bothcarboxylate and carbonate groups, thus, for example,acyloxy substituted alkyl groups include for example t li 012339 - 22 - alkylcarbonyloxy alkyl. In such groupa any alkylenemoieties preferably hâve carbon atom contents definedfor alkyl groupe above. Preferred aryl groupe includephenyl and monocyclic 5-7 membered heteroaromatics,especially phenyl and such groups may themselvesoptionally be substituted.

Représentative substituted alkyl groups Rx includealkoxyalkyl, hydroxyalkoxyalkyl, polyhydroxyalkyl,hydroxy poly alkyleneoxyalkyl and the like such asalkoxymethyl, alkoxyethyl and alkoxypropyl groups oracyloxymethyl, acyloxyethyl and acyloxypropyl groups eg.pivaloyloxymethyl. f

As used herein substituted groups may be mono orpoly substituted by hydroxy, alkoxy, acyloxy, nitro,alkoxycarbonyloxy, amino, aryl, oxo or halo groupsunless specifically stated otherwise.

In another preferred embodiment of the présentinvention, the COX-2 inhibitor is selected frommodifications of classical NSAID's, for example the pro-drugs, esters or salts thereof.

With basis in the Chemical structures of classicalNSAIDs, more new sélective COX-2 inhibitors hâve beenprepared. Such a compound may be mêloxicam which is anoxecam (the COX-2 spécifie analogue of the well knownpiroxicam), or acetic acid dérivatives such as etodolac(COX-2 spécifie analogue of diclofenac). Other examplesof some of the most preferred compounds in this classare COX-2 active indomethacin dérivatives and zomepirac. A further listing of families and subfamilies ofcompounds according to the présent invention is found in-patents and patent applications on COX-2 inhibitors; forexample in the patent documents previously listed inthis text. These patent documents also exemplify andlist spécifie compounds that also are the most preferredCOX-2 inhibitors according to the invention.

Particularly preferred compounds are however:diisopropylfluorophosphate, L-745337, rofecoxib, NS 398, 012339 - 23 - SC 58125, etodolac, meloxicam, celecoxib and nimesulide.

Methods for producing COX-2 inhibitors for use inaccordance with the invention are well known to those inthe art, particularly as described in the literature 5 mentioned above. A COX-2 inhibitor for use in treatment andprévention of disorders as described herein, e.g.immunodeficiencies and viral infections, especiallyHIV/AIDS, according to the présent invention may contain 10 one or more asymmetric centres and/or one or more doublebonds—i.e. the' invention extends to use of isomers andracematçs of the compounds disclosed herein. Ail suchpossible isomers are within the scope of the présentinvention. The COX-2 inhibitor can be in the form of an 15 isomeric mixture of compounds or more preferably in theform of a purified isomer or a pharmaceuticallyacceptable sait thereof.

The pharmaceutical composition of COX-2inhibitor(s) for treatment of conditions according to 20 the invention, e.g. immunodeficiencies and viralinfections can be formulated as pharmaceuticallyacceptable salts and can also contain pharmaceutically - acceptable carriers well known in the art.

Thus, the présent invention also extends to 25 pharmaceutical compositions comprising a COX-2 inhibitoror dérivative or pharmaceutically acceptable saitthereof and a pharmaceutically acceptable diluent,carrier or excipient. By "pharmaceutically acceptable" ‘ is meant that the ingrédient must be compatible with 30 other ingrédients in the composition as well asphysiologically acceptable to the récipient.

In further embodiments the présent invention alsoextends to the use of such compositions and methods ofprévention/ treatment using such compositions, as 35 described hereinbefore.

If the COX-2 inhibitor is basic, salts can beprepared from pharmaceutically acceptable non-toxic U12339 - 24 - acids including inorganic and organic acids.

Particularly preferred salts are hydrochloric, hydrobromic, phosphoric, sulfuric, ci trie, maleic,citric'and tartaric acid salts.

If the COX-2 inhibitor is acidic, salts can beprepared from pharmaceutically acceptable non-toxicbases including inorganic or organic bases.

Particularly preferred salts are sodium, potassium andmeglumine salts.

For the treatment and prévention of disorders as -described herein, e.g. immunodeficiency or virarldiseases including HIV/AIDS, the COX-2 inhibitors, can beadministered orally, rectally, topically, buccally, byinhalation or parenterally (e.g. intramuscularly,subcutaneously, intraperitoneally or intravenously) inthe form of an injection or infusion. The preferredadministration forms will be administered orally,rectally and by injection or infusion. The mostpreferred administration form will be suitable for oraladministration.

For ail administration forms, the COX-2 inhibitoris administered in dosage unit formulations usuallycontaining well known pharmaceutically acceptablecarriers, adjuvants and vehicles. Thus, the activeingrédient may be incorporated, optionally together withother active substances as a combined préparation, withone or more conventional carriers, diluents and/orexcipients, to produce conventional galenic préparationssuch as tablets, pills, powders, lozenges, sachets,cachets, élixirs, suspensions, émulsions, solutions,syrups, aérosols (as a solid or in a liquid medium),ointments, soft and hard gelatin capsules, suppositories, stérile injectable solutions, stérilepackaged powders, and the like. Biodégradable polymers(such as polyesters, polyanhydrides, polylactic acid, orpolyglycolic acid) may also be used for solid implants.The compositions may be stabilized by use of freeze- 012339 - 25 - drying, undercooling or Permazyme.

Suitable excipients, carriers or diluents arelactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, aglinates, 5 tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water syrup,water, water/ethanol, water/glycol, water/polyethylene,glycol, propylene glycol, methyl cellulose,methylhydroxybenzoates, propyl hydroxybenzoates, talc, 10 magnésium stéarate, minerai oil or fatty substances sûchas hard fât or,' suitable mixtures thereof. Thecomposition^ may additionally include lubricatingagents, wetting agents, emulsifying agents, suspendingagents, preserving agents, sweetening agents, flavouring15 ' agents, adsorption enhancers, e.g. for nasal delivery(bile salts, lecithins, surfactants, fatty acids,chelators) and the like. The compositions of theinvention may be formulated so as to provide quick,sustained or delayed release of the active ingrédient20 after administration of the patient by employingprocedures well known in the art.

The active ingrédient for administration may beappropriately modified for use in a pharmaceuticalcomposition. For example, the active ingrédient may be25 stabilized for example by the use of appropriate additives such as salts or non-electrolytes, acetate,SDS, EDTA, citrate or acetate buffers, mannitol,glycine, HSA or polysorbate.

Conjugates may be formulated to provide improved30 lipophilicity, increase cellular transport,, increase solubility or allow targeting. These conjugates may becleavable such that the conjugate behaves as a pro-drug.Stability may also be conferred by use of appropriatemétal complexes, e.g. with Zn, Ca or Fe.

The active ingrédient may be formulated in anappropriate vehicle for delivery or for targetingparticular cells, organs or tissues. Thus the 35 h 012339 - 26 - pharmaceutical compositions may take the form ofmicroemulsions, liposomes, niosomes or nanoparticleswith which the active ingrédient may be absorbed,adsorbed, incorporated or bound. This can effectivelyconvert the product to an insoluble form.

These particles may carry appropriate surfacemolécules to improve circulation time (e.g. sérumcomponents, surfactants, polyoxamine908, PEG etc.) ormoieties for site-specific targeting, such as ligands toparticular cell borne receptors, Appropriate techniquesfor drug delivery and for targeting are well known -xnthe art, but see for example Kreuter, 1994, Eur. J. DrugMetab. Pharmacokinet., 3, p253-256; Shen, 1997, J. DrugTargeting, 5(1), pll-13; Mrsny, 1997, J. Drug Targeting,5(1), p5-9; Pettit &amp; Gombotz, 1998, TIBTECH, 16, p343-349; and Duncan, 1997, J. Drug Targeting, 5(1), pl-4regarding drug targeting and Simari &amp; Nabel, 1996,

Semin. Intervent. Cardiol., 1, p77-83; Torchilin, 1998,J. Microencapsulation, 15(1), pl-19; Klyashchitsky &amp;Owen, 1998, J. Drug Targeting, 5(6), p443-458; Kreuter,1996, J. Anat., 189, p503-505; Fasano, 1998, TIBTECH, 16, P152-157; Kataoka et al., 1993, 24, pll9-132;Anderson, 1998, Nature, 392(suppl), p25-30;-Langer, 1998, Nature, 392(suppl), p5-10; Gregoriadis, 1995,TIBTECH, 13, Ρ527-536; Gregoriadis et al., 1997, FEBSLett., 402, pl07-110; Rolland, 1998, Critical Reviews inTherapeutic Drug Carrier Systems, 15(2), pl43-198; Hopeet al., 1998, Molec. Memb. Biol., 15, pl-14; andScherman et al., 1998, Curr. Opinion Biotech., 9(5),p480-485 regarding peptide and nucleic acid moléculedelivery. For an example of spécifie site directedtargeting, see for example Schâfer et al., 1992, Pharm.Res., 9, p541-546 in which nanoparticles can beaccumulated in HIV-infected macrophages. Clearly suchméthode hâve particular applications in the methods ofthe invention described herein.

Such derivatized or conjugated active ingrédients 012339 - 27 - are intended to fall within the définition of inhibitorymolécules which are used according to the invention.

Thus for example, the pharmaceutical compositionfor oral use contains the active ingrédient (s) andsuitable physiologically acceptable agents to formtablets, capsules, solutions, suspensions or other wellknown formulations for oral administration. Suchcompositions can be prepared according to any methodknown for the manufacture of oral pharmaceuticalcompositions. Such compositions can contain one or morebiologically active agents and one or more agentsselected from the group of preserving agents, inertdiluents, viscosity increasing agents, colouring agents,sweetening agents, granulating agents, disintégrâtingagents, binding agents, osmotic. active agents, wettingagents, suspending agents, matériels for préparation ofdelay formulations, oils and water.

Pharmaceutical compositions for other than oraluse, for example suppositories for rectal administrationor solutions for injections or infusions can be preparedusing well known methods and additives for suchformulations. Ail formulations for injection andinfusion should be stérile formulations.

The active ingrédient in such compositions maycomprise from about 0.01% to about 99% by weight of theformulation, preferably from about 0.1 to about 50%, forexample 10%.

For treatment of disorders in accordance with theinvention, e.g. immunodeficiencies and viral infections,with COX-2 inhibitors, the dose levels per day are inthe range 0.005 mg to about 150 mg/kg of body weight.

The dose dépends strongly on the choice of the COX-2inhibitor compound, the clinical situation (type ofvirus, status of the infection and condition of thepatient) , the patient's âge and weight, route ofadministration and the total use of drugs by the patientincluding the length of the course of treatment. More 012339 - 28 - preferred doses will normally be between 0.01 mg and 50æg/kg of body weight daily, and even more preferably0.05 mg to 20 mg/kg of body weight daily. Thus forexample, 25 mg of rofecoxib or 200 mg celecoxib may beadministered daily by oral administration to an adulthuman.

Dosage units are generally between 1 mg and 500 mg.of the active ingrédient.

According to one aspect of the présent invention,one COX-2 inhibitor can be combined with one or moreother COX-2 inhibitors to treat disorders as described *“herein, e.g. an immunodeficiency or viral infection.

According to another aspect of the présentinvention, the COX-2 inhibitor can be combined with oneor more further COX-2 inhibitors or one or more otherdrugs with different modes of action to treat thedisorder, e.g. the immunodeficiency, HIV infection, orAIDS. Examples of such combinations could be COX-2inhibitor in combination with one or more NNRTIs or incombination with one or more NRTIs or in combinationwith one or more HIV protease inhibitors or one or moreHAART in combination with the COX-2 inhibitor.

In a further aspect the présent invention providesmethods and/or compositions which combine one or moreCOX-2 inhibitors with compounds that improve thetolerability of the active ingrédient, especially duringlong term treatment. Typical compounds includeantihistamine and proton pump inhibitors.

Thus the invention extends to a compositioncomprising a COX-2 inhibitor as described hereinbeforetogether with one or more additional COX-2 inhibitorsand/or one or more additional active ingrédients. Theinvention further extends to use of such compositionsand methods of using such compositions as describedhereinbefore. The invention further extends to aproduct comprising the components described above as acombined préparation for simultaneous, separate or 012339 - 29 - sequential use in treating or preventing conditions ordisorders as described hereinbefore.

The invention is further described in the followingnon-limiting Examples with reference to the followingFigures :

Figure _1 shows cyclic AMP levels following MAIDSinfection in CD8+ (A), CD4 + (B) and B (C) cells.Mononuclear cells were isolated from lymph nodes of miceinfected with MAIDS for varions période of time andseparated intg' CD4-F7 CD8+ and B cells by négativesélection using a FAGS-cell sorter. Intracellular cAMPlevels were assessed by sonication and radioimmunoassay.Bars represent mean ± SD (n=3 individual mice) ;

Figure 2 shows MAIDS cAMP levels in CD4+, Thy-1.2négative and positive populations. Lymph node cellsfrom three infected and three age-matched control micewere FACS-sorted into CD4+, Thy-1.2+ (open bars) andCD4+, Thy-1.2- (solid bars) populations, andintracellular cAMP levels were assessed as in Figure 1.Bars represent mean ± SD (n=3) ;

Figure 3 shows levels of protein kinase A activity inMAIDS vs wild type mice.' (A) Kinase activities usingKemptide as substrate in the presence (total activity,hatched bars) or absence (free activity, solid bars) of5 μΜ cAMP was examined in detergent-solubilized extractsof lymph node cells purified from mouse spleens.Phosphotransferase activity not inhibited by the PKA-specific protein kinase inhibitor (PKI, 1 μΜ) wassubtracted to show only the ΡΚΆ-specific activity.Activities in infected mice (MAIDS; n=4) are shownrelative to those of wild type littermates. (B) [3H- cAMP] binding was measured in the same extracts as in(A) , and molar amounts of R monomer were calculâted;Figure 4 shows immunolocalization of PKA C-subunit incells of MAIDS and wild type mice. Mononuclear cellsfrom control mice (upper panel) and mice infected with I»

I 012339 - 30 - MAIDS (two lower panels) were attached to glass slidesby cytospin (400 x g) , fixed and immunostained withanti-PKA-C polyclonal antibody and HRP-conjugatedsecondary antibody (brown stain). Counterstaining is byhematoxylin (blue stain on chromatin) ;

Figure 5 shows the effect of the PKA type I antagonistRp-8-Bromo-cAMP-phosphorothioate (Rp-8-Br-cAMPS) on Tcell function in MAIDS and wild type mice. TCR/CD3stimulated T cell prolifération was assessed withisolated T cells from MAIDS mice (A) and uninfectedcontrol mice (B). The effect of increasing concentrations of cAMP agonist (8-CPT-cAMP) on TCR/CD3stimulated prolifération of CD3+ T cells isolated fromMAIDS (open circles, dotted line) and control mice(filled circles and solid line) was examined separatelyin the same expérimente (C) . Mean values of triplicatedéterminations ± SD are shown. See Table 4 forsummarised data (n=ll). Note: Scaling differs in A andB, whereas in C the TCR/CD3 induced prolifération in theabsence of cAMP agonist is normalized to 100% for bothMAIDS and control T cells;

Figure 6 shows sécrétion of PGE2 by normal and MAIDSlymph node cells in vitro. Unsorted lymph node cellsfrom MAIDS infected mice (solid bars, n=9) at 20 weekspost infection and age-matched control mice (shadedbars, n=4) were cultured for 48 h in complété mediumafter which secreted levels of PGE2 were measured in thesupernatants by ELISA;

Figure 7 shows the effect of a non-sélective COXinhibitor on T cell immune function in normal and MAIDSinfected mice. Column 1 - control mice + anti-CD3;column 2 - control mice + anti-CD3 + indomethacin;column 3 - MAIDS mice + anti-CD3; column 4 - MAIDS mice+ anti-CD3 + indomethacin. T cell proliférativeresponses were assessed in a mixed population ofunsorted lymph node mononuclear cell by [3H] - thymidineincorporation in the absence and presence of the non- 012339 - 31 - sélective COX inhibitor indomethacin (50 ng/ml). T cellactivation was accomplished by cross-ligation of anti-CD3 (mAb 2C11; 4 /ig/ml) . Bars show mean values ± SDfrom control (n=3) and MAIDS infected (n=5) mice, seeTable 5 for additional data. Celle were cultured for 72h during which [3H] - thymidine was included for the last 4h;

Figuxe-Æ shows expression of COX-2 by different subsetsof lymph node lymphocytes in normal (A) and MAIDSinfected (B) mice. CD4+ T, CD8+ T and B cells wereFACS-sorted by' positive-selection on basis of expressionof the CD4, CD8 and B220 jnolecules, respectively. CDllb- cells were sorted by négative sélection (on thebasis of absence of CDllb) . Cells from MAIDS infectedand normal mice were then lysed and 10 μ<3 of proteinfrom each sample were subjected to immunoblot analysisfor the expression of COX-2. Blots were concomitantlyreacted with antibodies to actin as control;

Figure 9 shows expression of CDllb in MAIDS and wildtype lymph node cells. Expression of CDllb (by flowcytometry) by the different subsets of lymph nodelymphocytes (CD4+, CD8+ T cells and B220+ B cells) fromMAIDS infected and control mice is shown. RI: CDllbhigh; R2: CDllb dim and R3: CDllb-.;

Figure 10 shows levels of expression of COX-2 in lymphnodes of MAIDS infected mice and wild type mice. Lymphnodes were freeze-sectioned and subjected to COX-2immunohistochemical staining (brown stain)'. (a) Normalcontrol lymph node with germinal center stained for COX- 2. (b) Normal lymph node at higher magnification.

Cells staining positive for HRP-colour reaction are"tingible body" macrophages with ingested material(arrows). c. Lymph node from MAIDS infected mouse (week20 post infection). Note: altered morphology andarchitecture, d. Higher magnification of MAIDS lymphnode stained for COX-2. Note: number of cells brownimmunostaining in the cytoplasra and numerous mitotic h u(zû oy - 32 - figures; .Figure—11. shows the effect of in vivo administration ofa non-selective COX inhibitor on T cell immune functionof HIV infected patients. T cell proliférativeresponses were assessed in CD3+ T cells as [3H]-thymidineincorporation from 3 patients (pat. 1 to 3) participating in a phase II clinical trial and receivingindomethacin 25 mg three times a day perorally for 14days in addition to triple combination therapy. Upperpanels shows T cell immune function at day 0, day 14(after 2 weeks treatment) and at day 28 (2 weeks afterdiscontinuâtion), labelled respectively as colurans 1, 2and 3. T cell activation was accomplished by cross-ligation of anti-CD3 (mAb SpVT3b) . A: Basal prolifération after T cell activation; B: proliférationin presence of Rp-8-Br-cAMPS (1 mM) ; Note: degree ofcAMP-mediated immunodeficiency is évident from comparingupper and lower panel. Bars show mean values ± SD fromtriplicate déterminations. Cells were cultured for 72 hduring which [3H] - thymidine was included for the last 16h;

Figure 12 shows the effect of in vivo administration ofa non-selective COX inhibitor indomethacin on T cellprolifération of HIV infected patients as described inFigure 11 but for 7 patients, indicated for patients 1to 7, respectively by filled circles, open circles,filled triangles, open triangles, filled squares, opensquare and filled diamonds. Mean values from triplicatedéterminations are plotted, connector Unes showdevelopment of each patient;

Figure 13 shows the effect of rofecoxib, a COX-2 spécifie inhibitor, on T cell immune function in MAIDSinfected mice. , T cell proliférative responses wereassessed in a mixed population of unsorted lymph nodemononuclear cells by [3H]-thymidine incorporation in theabsence and presence of increasing concentrations (1.9to 500 nM) of the COX-2 spécifie inhibitor, rofecoxib. 012339 - 33 - T cell activation was accomplished by cross-ligation ofanti-CD3 (mAb 2C11; 4 gg/ml) . Mean values fromtriplicate déterminations are shown together with asigmoid curve fit. Cells were cultured for 72 h duringwhich [3H] -thymidine was included for the last 4 h;

Figure 14 shows the effect of celecoxib, a COX-2spécifie inhibitor, on T cell immune function in MAIDSinfected mice, as described in Figure 13 for rofecoxib;Figure 15 shows the effect of rofecoxib and celecoxibcompared to indomethacin on the sécrétion of PGE2 bylymph node (LNj cells ex vivo for control mice (1) orMAIDS mice (2) . Unsorted LN cells were cultivated incomplété medium in the presence or absence of the PGE2inducer, lipopolysaccharide.. {LPS; 4 μg/ml) ; thenonspecific cyclooxygenase inhibitor, indomethacin (50ng/ml);and the COX-2 spécifie inhibitors rofecoxib(0.125 μΜ) and celecoxib (0.125 μΜ) . After 48h, theconcentration of PGE2 was measured by EIA in thesupernatants. 3 individual infected mice (week 20) and pool of 3 age-matched Controls were analyzed. Means ±standards déviations are shown; and

Figure 16 shows the. effect of in vivo treatment of MAIDSmice with rofecoxib- on T cell immune function. MAIDSmice were left untreated (untreated 1 to 3) or treatedwith rofecoxib per os (3 mg/kg/day administered oncedaily, treated 1 and 2) for seven days administered viaa tube inserted in the ventricle. Subsequently, T cellproliférative responses were assessed in vitro in amixed population of' unsorted lymph node raononuclearcells from treated and untreated animais by- [SH] -thymidine incorporation in the absence (columns A) andpresence of Rp-8-Br-cAMPS (0.5 or 1.0 mM, columns B andC, respectively). T cell activation was accomplished inail samples by cross-ligation of anti-CD3 (mAb 2C11; 4μρ/πιΐ) . Control représenta T cell prolifération inuninfected mice. Mean values from triplicatedéterminations are shown. Cells were cultured for 72 h 012339 - 34 - during which [3H]-thymidine was included for the last 4hour s; E.jgure 17 shows the effect of. in vivo treatment of MAIDSmice with rofecoxib or celecoxib on T cell immune 5 function. MAIDS mice were injected with vehicle (intralipid) , treated with rofecoxib in intralipid byintraperitoneal injection (3 mg/kg/day administered oncedaily, n=6) or treated with celecoxib by intraperitonealinjection (20 mg/kg/day administered once daily, n=5) 10 for 18 to 20 days. Subseguently, T cell proliférative. ·responses were assessed in vitro as described for Figure16 but without Rp-Br-cAMPs. Control represents T cellprolifération in uninfected mice. Mean values fromtriplicate déterminations are shown (black circles) 15 along with 25 to 75% percentile (boxed areas) and médian(line in box). ' Bars represent range/ andFigure 18 shows the effect of in vivo treatment of MAIDSmice with meloxicam on T cell immune function. Osmoticpumps (Alzet, 100 μΐ) with meloxicam (release rate of 70 20 ^g/animal/day) or phosphate buffered saline (PBS) wereimplanted subcutaneously on MAIDS mice (14 weeks postinfection) and healthy mice for 14 days. a),Subsequently, T cell proliférative responses wereassessed in vitro as described for Figure 17. Mean 25 values ± standard error of the mean (s.e.m.) from eachgroup are shown. The effect of meloxicam treatment onanti-CD3 stimulated prolifération of cells from MAIDSmice (solid bars) compared to that of MAIDS mice thatreceived PBS (open bars) is significant (p<0.05). b), 3 0 Mixed lymph node cultures from the groups of mice in a) treated in vivo with meloxicam or PBS were added backmeloxicam (2.5 ^g/ml) in cell culture in vitro, anti-CD3induced T cell prolifération was assessed as in a) , andthe effect of meloxicam added back in vitro (open bars) 35 was compared to the response of the cells with no invitro addition (solid bars) (p=0.005). c),

Rp-8-Br-cAMPS (0.5 mM) was added to in vitro cell 012339 - 35 - cultures of mixed lywph node cultures from the groups ofmice in a) treated in vivo with meloxicam or PBS,anti-CD3 induced T cell prolifération was assessed as ina), and the effect of Rp-8-Br-cAMPS in vitro (open bars) 5 was expressed as fold induction above that of celle thatreceived no in vitro addition (solid bars). Statisticswere analysed by Mann-Whitney U. test for. comparison oftwo groups of animais and with Wilcoxon Matched PairsTest for comparison of the saine group with two different 10 treatments. » t 012339 - 36 -

EXAMPLES EXAMPLE 1

Mi.ce...wi.th· murine .accuired immunodeficiencv syndrome (MAIDS) hâve a cAMP/PKA type I induced T cell dysfunction MAIDS (Murine Acquired Immunodeficiency Syndrome).Numerous studies hâve considered MAIDS as a possiblemodel for infection of humans by HIV. This syndromedevelops following infection with a replication-defective retrovirus that encodes a variant Pr609®9polyprotein (Chattopadhyay et al., 1991, J. Virol., 65,p4232-4241; Jolicoeur, 1991, FASEB J., 5, p2398-2405).

The syndrome is associated with progressive lymphoprolifération in the spleen and lymph nodes andsevere immune defects. Although the defectiveretrovirus responsible for MAIDS infects mostly B cells(Aziz, 1989, Nature, 338, p505-508), CD4+ T cells displaya profound dysfunction and anergy to mitogen stimulationin vitro. A large fraction of 004* T cells (but not CD8+T cells) of infected mice are also characterized by anunusual Thy-1 négative phenotype (Holmes et al., 1990,Eur. J. Immunol., 20, p2783-2787; Moutschen et al., 1994, Scand. J. Immunol., 39, p216-224 (MAIDS)). Innormal, uninfected mice, CD4+ Thy-1' T cells are foundselectively in the germinal centers where theycorrespond to recent antigen-spécifie émigrants.

The mechanism by which the variant Pr609a9 proteininduces T cell abnormalities is not known. Solublefactors secreted by infected cells hâve been claimed toinfluence the function of T cells (Simard, J. Virol., 68, pl903-1912) at a distance, but the nature of suchmediators has never been elucidated. Other studies hâvesuggested that direct, cognate interactions between CD4+ T cells and antigen presenting cells are necessary for 012339 - 37 - the induction of T cell defects (Green, 2001, J. Virol.,70, p2569-2575; de Levai, 1998, J. Virol., 72, p5285-5290.

The adenylate cyclase-cAMP-protein kinase A pathwayplays an important rôle in the régulation of immuneresponses (Kammer, 1991, Immunol. Today, 9, p222-229) .Increased concentration of cAMP is known to inhibitproliférative responses of T cells to various stimulisuch as anti-CD3 mAb and interleukin-2. A recent reporthas suggested that downregulation o£_the JAK3 tyrosinekinase might represent a raechanism by which cAMPinhibits T cell prolifération (Kolenko7 1999, Blood, 93,p2308-2318). Cyclic AMP could also induce thedownregulation of membrane proteins since murinethymocytes or thymoma cells exposed to cAMP inducingagents such as norepinephrine downregulate Thy-1expression by a mechanism involving destabilization ofmRNA (Wajeman-Chao, J. Immunol., 161, p4825-4833) .

Prostaglandin E2 (PGE2), a potent inducer of cAMP, ismainly secreted by monocytes, macrophages and activatedT cells. PGE2 shifts the balance from T-helper type 1cells toward T-helper type 2 cells by inhibiting IL-2and enhancing IL-4 production (Betz and Fox, 1991, J.Immunol., 146, plO8-H3; Meyaard, 1997, Blood, 89, p570-576) . It also skews the différentiation of B cellstoward IgE production (Fedyk and Phipps, 1996, PNAS USA,93, pl0978-10983). Prostaglandin synthesis results fromthe sequential action of cyclooxygenase-1 and -2 (COX-1and COX-2) and spécifie PG synthases (Smith and DeWitt,1996, Adv. Immunol., 62, p!67-215). While COX-1expression is largely constitutive and ubiquitous, COX-2is only induced in certain cell types (macrophages,f ibroblasts', smooth muscle cells) by NO and inflammatorycytokines such as IL-1 and TNF-a. 012339 - 38 -

The mechanisms responsible for T cell dysfunction inMAIDS are still poorly understood. CD4* T celle arepreferentially involved whereas several reports hâvesuggested that the alteration of CD8+ T cells is only dueto the lack of adéquate CD4+ T cell help. In contrast,the inhibition of B cell responses is intrinsic andcannot solely be explained by defective CD4+ lymphocytes.The Inventors' observation of a sélective increase ofCAMP in B cells and CD4* T cells and not in CD8+ T cellsis therefore compatible with the involvement of cAMP inthe anergie process associated with MAIDS.

Z Tô" the Inventors1 knowledge, this is the first démonstration of a subset sélective increase of cAMP ina disease model. If a soluble factor such asprostaglandin E2 is indeed responsible for cAMPinduction, what could explain the subset selectivity ofits action? Former studies had comparecf the expressionof various prostanoid receptors on CD4+ and CD8+ T cellsand concluded a similar pattern of expression in bothsubsets. Normal CD8+ T cells are fully susceptible tothe cAMP inducing effects of PGE2. A possibleexplanation could take place at the post receptor level;memory/activated T cells are more responsive to PGE2 thannaïve T cells. In MAIDS, where MHC class II-dependentprocesses are involved, CD4+ T cells could acquire aparticular state of activation making them moresusceptible to the effect of a given concentration ofPGE2. Postreceptor modulation of prostanoid effects isprincipally mediated by G receptor kinases (GRK) whichuncouple protein G from the corresponding membranereceptor. Inflammatory States such as rheumatoidarthritis are characterised by a downregulation of GRKand therefore by an increased lymphocyte sensitivity tocAMP inducing agents such as catecholamines. Levels ofGRK activity in CD4+ and CD8+ T cells from infected miceis unknown. 012339 - 39 -

Methods used in Examples 1 and 2

Mice and cell suspension

Male C57BL/6 mice were bred in the Inventors' facility.Mice were injected twice i.p at the âge of 4 and 5 weekswith 0.25 ml of the cell f ree viral extract. Age-matched control mice were injected twice i.p. with 0.25ml phosphate buffered saline (PBS). At different timespost-infection, mice were killed by CO2 asphyxiation.Peripheral lym£>h nodes (inguinal, axillanty and cervical)were dissociated with syringes to obtain single cellsuspensions and passed through a nylon cell stainer,washed three times with RPMI 1640 complété medium andcounted on Thoma cytometer after trypan blue exclusion.

Virus

Viral extract was prepared from lymph nodes of miceinjected 2 months earlier with RadLV-Rs as describedpreviously. Lymph nodes were collected, ground in BBSand centrifuged at 1.5 x 104 g for 30 min. Thesupernatant was spun again for 30 min at 1.5 x 104 g.This acellular viral extract was-stored in liquidnitrogen. XC plaque assay was used to quantify theviral particles. The viral préparation contained 103particle forming units (PFU) ecotropic virus/ml.

Antibodies

The following polyclonal antibodies were used forwestern blotting experiments; Primary: polyclonal rabbitanti-COX-1 or rabbit anti-COX-2 antibody.(Santa CruzBiotechnology); Second-step: Horseradish PeroxidaseConjugated anti-rabbit was purchased from TransductionLaboratories (Transduction Laboratories, UK) . For theflow cytometry, the moAbs used are as follows: PE-conjugated CD4/L3T4 (YTS.191.1), FITC-conjugatedCD45R/B220 (RA3-6B2), FITC-conjugated GDIlb/Mac-1 012339 - 40 - (Ml/70), FITC-Conjugated CD161/NK-1.1 (PK136), FITC-conjugated CD8a (Ly-2) and CD16/CD32 (FcylII/XIReceptor) (2.4G2), (ail from Pharmingen: San Diego, CA,USA) . CD3 moAb (145-2C11) was purified in theInventors' laboratory. Concanavalin A (ConA) waspurchased from Boehringer Mannheim Biochemica andphytohemagglutinin-M (PHA) from Difco.

Flow cytometry and cell sorting

Analysis were performed by using FACStar-plus flow cellsortir with the Cellquest software (Becton Dickinson) .The forward and side scatters were used to gâte viablelymphocytes. For two-colour analysis of FITC (green)and PE (orange), blue excitation at 488nm was providedby an argon ion laser {Air-to-Water cooled modelSpinnaker 1161; Spectra Physics, Mountain View, CA) .

For cell sorting, 60 x 10® cells were incubated withanti-FcyRII (Fc Block) to prevent non spécifieinteractions, prior to labelling for 20 min on ice withthe f luorochrome-conjugated antibodies. CD4+ T cellswere negatively selected by depleting CD8+ B220+ CDllb+cells. Similarly, CD8+ T cells were negatively selectedby depleting CD4* B220+ CDllb* cells and B cells bydepleting CD8+ CD4+ CDllb* cells. For each sorting, theselected fraction was reanalyzed by flow cytometry toassess purity which was always higher than 97%.

Cyclic AMP quantitation

Single lymph node cell suspensions were prepared asdescribed above, washed twice with RPMI 1640 andcentrifuged at 1500 x g for 3 min. Cells weresubseguently disrupted by sonication to facilitate therelease of intracellular cAMP into the extractionsolution (0.01N HCl, 95% éthanol). The solutioncontaining the cell lysate was centrifuged at 13 x 10* xg for 15 min, and the supernatant was removed to a freshtube. The extract was evaporated in a Speed Vac 012339 - 41 - concentrator at 45°C, and the pellet was stored at -20°C.Just before use, the pellet was resuspended in the assaybuffer and cAMP levels were measured by radioimmunoassay(RIA) using 125I-Labelled cAMP assay System (Amersham,England) . The concentration of cAMP in test samples wasdetermined by comparison with a curvi-linear standardcurve. For positive and négative Controls,.lymph nodecells (l x 106) were incubated respectively with 1 mM ofdDibutyryl-cAMP and 0.5mM of DDA (Adenylyl cyclaseinhibitor) for 30 min at 37°C in a humidified 5% CO2 airincubator be foire measurement of cAMP concentration.

Z

Cell homogenization and immunoblotting

Cells (50 x 106) were homogenized by sonication (2 x 15s) on ice in a buffer containing 10 mM potassiumphosphate, pH 7.1, 250 mM sucrose, 1 mM EDTA, 0.1 %triton X-100 and 10 μg/ml each of the proteaseinhibitors chymostatin, leupeptin, pepstatin A and.antipain (Tasken et al, 1993, J. Biol. Chem., 268,P21276-21283) , and centrifuged for 30 min (15,000 x g)to remove unsoluble material. Protein concentrationswere determined by Bradford assays (BioRad) . Forimmunoblotting, 40 pg of protein was -separated by 10%SDS-PAGE, transferred to PVDF membranes and incubatedwith antibodies in TBS/Tween with 5% non-fat dry milkand 0.1 % BSA (Blotto) . Primary antibodies weredetected by HRP-conjugated secondary antibodies (JacksonLaboratories/Transduction Laboratories) and ECL(Amersham) .

Phosphotranaferase activîty of PKA

Catalytic activity of PKA was assayed by phosphorylâtinga PKA-specific substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly)(Kemp et al, 1976, PNAS USA, 73, pl038-1042) Kemptide,Peninsula Laboratories INC.) using [y-32P]-ATP (spécifieactivity 0.25 Ci/mMol, Amersham) in an assay mixturedescribed by R. Roskoski (Methods Enzymol,, 1983, 99, 012339 - 42 - p36) . phosphotransferase activity was measured both inthe presence and absence of cAMP (5 μΜ) and PKI (1 μΜ) ,and the low levels of activity not inhibited by PKI wassubtracted to détermine PKA-spécifie activity.

Cyclic AMP binding measurements

Quantification of spécifie [3H]cAMP binding of solubilized PKA regulatory subunits was performed asdescribed by Cobb and Corbin (Methods in Enzymology, 159, p202-208, 1988) in a mixture containing [2,8-3H1cAMP—(2.25 μΜ; spécifie activity of 5 Ci/mMol; DuPont-New England Nuclear) . Molar ratios of R subunitswere calculated based on two cAMP binding sites on eachregulatory subunit monoraer.

Immunocy tochemî b try

Control and infected lymph node lymphocytes were fixedwith cold acetone for 5 min and washed twice for 5 mineach in 0,1% of saponin in PBS. Endogenous peroxidasewas blocked by incubation with 0,3% hydrogen peroxide in0.1% saponin/PBS for 15 min. After rinsing insaponin/PBS, the si ides were incuba ted for 30 min at RTwith blocking buffer (1,5% normal goat sérum in 0,1%saponin / PBS) , followed by incubation for 60 min withprimary antibody solution at RT in a humidified chamber.Antibody against Ca was from Santa Cruz and was dilutedat 1:1000 in PBS containing 0,1% of saponin and 0,5% ofnormal goat sérum. Slides were then washed as beforeand incubated with biotinylated goat anti-rabbitantibody. This later was detected by ABC complex(Novastain Super ABC Kit, Novocastra). Peroxidase wasrevealed using diaminobenzidine (DAB) (Dako) which givesa brown precipitate in the presence of H2O2. Slides werecounterstained with hematoxylin-eosin (Sigma) . Thespecificity was tested by incubating the cytospin withspécifie peptide against the PKA-Co: subunit. 012339 - 43 -

Inrnunohi s tochemi s try

Immunohistochemistry was performed on 2,um-thin histological sections done in 4% paraformaldéhyde fixedand plastic embedded tissues (JB4-JBPolysciences).Sections were permeabilized with trypsin (0.24%) for 1min at 37°C, and then with Tween 20 (2%) for 30 min at37°C. Endogenous peroxidases were quenched byincubation with H2O2 (1%) for 30 min at room température.Aspecific sites were saturated with normal goat sérum(1.5%) during lh at 37°C. Sections were then incubatedovernight at with primary polyclonal rabbit ctnti-COX-1 or rabbit anti-COX-2 antibody (Santa Cruz ,Biotechnology) and then for 2h with biotinylated goatanti-rabbit antibody. This latter was detected by ABCcomplex (Novostain Super ABC Kit, Novocastra) .

Peroxidase was revealed using diaminobenzidine (DAB)(Dako) which gives a brown precipitate in the presenceof H202. Sections were counterstained with haematoxylin-eosin (Sigma) . The specificity was tested by incubatingsections with normal rabbit sérum instead of primaryantibody.

Prolifération assaya for MAIDS mice

Prolifération assays were performed by incubation of 0.1X 106 CD3+ T cells/ml in a 100 μΐ volume in fiat-bottom96-well microtiter plates. Activation was achieved bysubséquent addition of monodisperse magnetic beadscoated with sheep anti-mouse IgG (Dynal, cat. no. 110.02) at a cell:bead ratio of 1:1 followed by additionof anti-CD3 (clone 2C11) at a final dilution of 4 gg/mlfor the expérimenta shown. The optimal concentration ofantibody was titrated carefully in the initial setup andparallel experiments at several different dilutions ofantibody was always performed. Prolifération wasanalyzed by incubating cells for 72 hours during which (3H]-thymidine (0.4 /xCi) was included for the last 4hours and collected with a cell harvester (Skatron, 012339 - 44 -

Sterling, VA, USA) onto glass fiber filters.

Incorporated precursor was counted in a scintillationanalyzer (Tri-Carb, Packard, Meriden, CT, USA) . cAMPanalogs, when used, were added 30 min prior toactivation by addition of anti-CD3 antibodies. 8-CPT-cAMP was from Sigma (St. Louis, MO) and Sp- and Rp-8-Br-cAMPS were from BioLog Life Science Company (Bremen,Germany) and were ail dissolved to concentrations of 4to 10 mM in PBS and concentrations calculated using theextinction coefficients given by the manufacturer.Indomethacin—was dissolved in water and used at aconcentration pf 50 ng/ml. PGE2 détermination 500 μΐ of a 48h-culture superaatant of lymph node cellsfrom control and infected mice were pipetted into 1.5 mlpolypropylene tubes to which were added 500 μΐ ofwater : éthanol (1.4) and 10 μΐ of ice cold acetic acid.

The tubes were gently mixed and left for 5 min at roomtempérature. This was followed by centrifugation at2500 X g for 2min. The supernatants were collected andrun through Amprep C18 minicolumns, which had beenprimed with 2 column volumes of 10% éthanol. Thecolumns were then washed with 1 volume of H2O and 1column volume of hexane. PGE2 was then eluted with 2 x0.75 ml of ethyl acetate. The fractions were collectedand evaporated under nitrogen to dryness. Finally, eachfraction was reconstituted in 100μ1 of assay buffer andPGE2 'was assayed using Amersham EIA kit as recommended bythe manufacturer.

Statistical analyses

For comparison of two groups of individuals, the Mann-Whitney Ü test (two-tailed) was used. Coefficients ofcorrélation (r) were calculated by the Spearman=s ranktest. Statistical and curve fit analyses were performedusing Statistica (StatSoft Inc., Tulsa, OK) and Sigma

I h 012339 - 45 -

Plot (Jandel Corporation, Erkrath, Germany) softwarepackages, respectively. Results are given as médiansand 25th to 75th percentiles if not otherwise stated, p-values are two-sided and considered significant when<0.05. MAIDS infection leads to elevated cAMP in CD4+ T celle -Mice inoculated with a mixture of retroviruses known asRadLV-Rs that causes development of MAIDS, weresacrificed at ,tiifferent time points after infection, "andlymph node cells were sorted by négative sélection us in®a flow cytometer/cell sorter into pure B cells and CD4+and CD8+ T cells. Intracellular cAMP levels wereassessed in the different cell populations followinginfection. As can be seen from figure l, cAMP levelswere strongly increased (more than 20-fold) in CD4+ Tcells after a few weeks of infection. At later stages,B-cell cAMP levels also increased whereas only minorchanges were observed in CD8+ T cells. Furthermore,when CD4+ T cells were separated into Thy-1.2+ and Thy-1.2- cells by positive sorting, it was évident that themajor increase in cAMP levels was in Thy-1.2-· cells(figure 2, 6-fold). This normally low-abundantpopulation also displayed higher basal levels of cAMPthan compared to those of the Thy-1.2+ when bothpopulations were harvested from uninfected mice.

Examination of PKA phosphotransferase activity inpostnuclear supernatants from detergent solubilizedextracts revealed that the total levels of cAMP-dependent kinase activity was decreased in MAIDS lymphnode cells whereas minor changes in the activity wereobserved in the absence of cAMP (Figure 3A) . This isconsistent with a chronic activation and dissociation ofPKA leading either to dégradation of the C subunit or totranslocation of C. Assessment of cAMP binding (Figure 012339 - 46 - 3B) revealed no changes in total levels of PKA Rsubunits. Immunocytochemistry of lymph node cells fromMAIDS- and control mice revealed increased levels ofimmunoreactive PKA C subunit in the nucléus (Figure 4).This is again consistent with an activation of the cAMP-PKA pathway in MAIDS. PKA type I antagonist improves T cell prolifération ofMAIDS T cells ~

In order to examine the effect of elevated cAMP andactivation of PKA on inhibition of TCR/CD3-induced Tcell prolifération, we used a sulfur-substituted cAMPanalog (Rp-8-Br-cAMPS) working as a full antagonist forPKA type I (Gjertsen, Mellgren, et al. 1995 1665 /id}.Figure 5A shows that in T cells from MAIDS-infectedmice, TCR/CD3-stimulated prolifération was less than 10%of that of T cells from uninfected control mice (figure5B). Furthermore, when the effect of the PKA type Iantagonist was assessed in MAIDS T cells, we observed aconcentration-dependent increase in TCR/CD3-inducedprolifération that was more than 4-fold at higherconcentrations (Figure 5A), whereas no stimulation wasobserved'by treatment of control T cells (Figure 5B).Looking at eleven MAIDS-infected mice, they ail hadseverely impaired T cell prolifération compared toControls (p<0.001) and in 10 out of 11 mice, the PKAtype I antagonist improved T cell prolifération (pcO.Ql;médian 2.2-fold, Table 5). The stimulatory effect ofthe cAMP-antagonist was not saturated even at thehighest concentrations used (Figure 5A and similar data(not shown) were obtained for ail mice in Table 5).

This indicates that the solubility of the compound,affinity, or availability to cells may be a limitingfactor for the effect observed. Thus, a more permeableand potent PKA type I antagonist, when available, mayfurther improve TCR/CD3-induced prolifération of MAIDS Tcells. b 012339 - 47 -

Next, the effect of cAMP agonist on TCR/CD3-inducedprolifération was investigated in five MAIDS-infectedmice and four Controls. T cells from MAIDS-infectedmice revealed an apparent shift in sensitivity toinhibition of cell prolifération by exogenously added 8-CPT-cAMP (Figure 5C and Table 5) . Moreover, when themaximal prolifération rates of T cells from MAIDS-infected mice and that of control T cells werenormalized to 100¾ (Figure 5C and data not shown), itwas évident that in addition to a left-shifted cAMP-inhibition cwçÿe, the slopes of the curves were " significantly different (Hill coefficients of 0.6 (0.54 / to 1.52) for T cells from MAIDS mice versus 2.2 (1.9-2.5) for normal T cells, Table 5, p<0.05). Theincreased sensitivity to inhibition by cAMP analogsuggests a contribution from elevated endogenous cAMP inpriming cAMP binding site B of PKA type I withsubséquent increase in the affinity of the A site forthe exogenously added cAMP analog. The shift in curveslope from a cooperative, two-ligand site bindingsituation to an apparent non-cooperative inhibitioncurve by 8-CPT-cAMP also indicates B-site occupancy byelevated endogenous cAMP. 012339 - 48 -

Table 5. 5 Mice Anti-CD3-induced prolifération (cpm) Increase inprolifération byRp-8-Br-cAMPS(fold increase) Inhibition ofprolifération by8-CPT-cAMP (ICjo, μΜ) Inhibition ofprolifération by 8-CPT-cAMP(Hill coefficient) 10 1 9525 19 6 41 2 3312 24 22 54 3 9153 14 8 58 4 959 37 n.d. n.d. 15 5 13791 "" 10 52 156 6 6370 19 66 152 7 6357 ~ 22 n.d. n.d. 8 9986 42 n.d. n.d. 9 5696 40 n.d. n.d. 20 10 16132 37 n.d. n.d. 11 3740 37 n.d. n.d. MAIDS 6370* 2,2** 0,22 0,58*** Médian (3740 - 9986) n=l 1 (1,9-3,7) (0.08-0,52) (0,54-1,52) 25 (25-75th n=ll n=5 n=5 percentiles) Controls 62281 1,1 0,40 2,24 30 Médian (56539 - 82038) (1,0-1,3) (0,33-0,46) (1,93-2,47) (25-75th n=6 n=6 n=4 n=4 percentiles) • MAIDS vs. Controls; * dénotés p<0,001, ** dénotés p<0,01 and *** dénotés p<0,05 35 Cvclic AMP-induced T cell dvsfunction of MAIDS is due to increased ..PGEo production bv CDllb-oositive cells with 40 increased levels of COX-2

Eleva ted production, of PGE2 in MAIDS -

Mixed lymph node cell populations were isolated from MAIDS-infected and control mice and cultured in vitro. 45. Secreted levels of PGE2 were assessed in media supernatants after 48 hours of culture and revealed that MAIDS infected cells secreted 7 to 8-fold more PGE2 than control cells. 0 72339 - 49 -

Inhibition of PGE2 production restores the T cell prolifération in MAIDS -

Next, mixed lymph node cells were activated by anti-CD3 antibodies to induce prolifération of T cells, and [3H]-thymidine incorporation was examined after 72 hours.Prolifération of cells from MAIDS-infected mice wasagain only 10 to 20 % of the T cell prolifération ofuninfected cells. However, when indomethacin was addedto the cultures to inhibit production of PGE2 in themixed cultures, this strongly increased the prolifération bf cells from five MAIDS-infected mice to " levels comparable to that of control mice (Figure 6) . x.

Looking at 10 additional MAIDS-infected mice (Table 6) , the effect of indomethacin on T cell prolifération of mixed lymphocyte cultures was very significant (p<0.01).

In contrast, treatment of control cultures withindomethacin did not alter prolifération. COX-2 is expressed at high levels in lymph nodes of MAIDS infected mice -

The constitutively expressed COX-1 is the normal source of cyclooxygenase activity that produces PGE2. However,no increase in COX-1 could be found in MAIDS mice thatcould account for the increased levels of PGE2 (data notshown). Expression of COX-2 is normally restricted tobrain/brain processes, to arthritic synovia and sites oftissue injury. COX-2 is not found in lymph nodes orlymphocytes as shown e.g. for control lymphocytes inFigure 8 (upper panel). Surprisingly, we found thatcrude lymph node cells from MAIDS infected mice expresshigh levels of COX-2 (figure 8, lower panel).

Furthermore, positively selected CD4+ and CD8+ T cellsas well as B cells from MAIDS lymph nodes contained highlevels of COX-2. In contrast, negatively selectedCDllb- cells contained only low levels of COX-2.

From looking at CD4+ and CD8+ T cells and B cells (B220 012339 - 50 - marker) from MAIDS infected and control mice by flowcytometry, it was évident that the CDllb marker is notnormally expressed on T or B celle. However, a distinctfraction of both CD4 + T cells and B cells from MAIDSinfected mice were CDllb bright (gating labelled RI) andan additional pool of CD4 + T cells and B cells as wellas CD8+ T cells were CDllb dim (gating labelled R2) ,indicating that they had significant but lower levels ofCDllb expression. Thus, subpopulations of MAIDS-infected CD4+ and CD8+ T cells were CDllb bright anddira, respectively, whereas the majority of B cells werepositive. Taken together vp.th the fact that CDllb+cells, and not CDllb- cells, expresses COX-2, thisindicates that both B cells and T cells in lymph nodesfrom MAIDS-infected mice express COX-2.

From looking at intact lymph nodes from MAIDS-infectedmice by immunohistochemistry, it is clear that the .grossarchitecture is altered with loss of germinal centers inMAIDS (week 19 post infection) compared to control mice(Figure 10, c versus a) . At higher magnification ofslides immunostained for COX-2, it is évident thatwhereas lymph nodes from control animais only show brownHRP-staining in the ingested material in macrophages(falsely positive "tingible" bodies, Figure 10b), alarge proportion of lymph node cells in MAIDS stainpositive for COX-2 (Figure lOd) .

I 012339 - 51 -

Table 6.

Mouse Medium Indomethaci n Anti-CD3 Indomethacin ZAnti-CD3 1 1304 1412 6245 9381 2 1082 1129 8019 47926 3 209 265 918 1345 4 236 335 8938 11579 5 4715 . 4317 6591 8545 6 1799 ND 2932 ND 7 3051 ND 7436 ND 8 1668 ND 3594 19624 9 839 2363 7885 31830 10 3413 7316 8777 42244 Médian 1486 1412 7013 15601" (25-75th (839- (335-4317) (3594-8019) (8963-37037) percentiles) 3051) n=10 ne7 .. n=10 n=8

Indomethacin (Indo) vs. Controls; ** dénotés p<0,0l EXAMPLE 3’ HIV patients exhibit marginal effects when.treated with non-selective COX inhibitor in vivo

Methods Négative sélection of peripheral blood CD3+ T celle front .HIV patients

Peripheral blood CD3+ T cells were purified by négativesélection from buffycoats from normal healthy donors(Ullevaal University Hospital Blood Center, Oslo,

Norway). Briefly, peripheral blood mononuclear cellswere isolated by density gradient (Lymphoprep, NycoMed,Oslo, Norway) centrifugation followed by négativesélection using monodisperse magnetic beads directlycoated with antibodies to CD14 and CD19 and rat anti-raouse IgG beads coated with antibodies to CD56 and amagnet. Magnetic beads were ail from Dynal (Oslo,

Norway, cat. no. 111.12, 111.04, and 110.11,respectively) whereas anti-CD56 antibody was from 012339 - 52 -

Pharmingen (San Diego, CA, cat. no. 31660.d ). Ailsteps were performed at 4 °C. Cell suspensions wereanalyzed by flow cytometry and shown to consist of morethan 90 % CD3+ cells.

Prolifération aasays using HIV patient T cells

Prolifération assays were performed by incubation of0.75 X 106 CD3+ T cells/ml in a 100 /il volume in flat-bottom 96-well microtiter plates. Activation wasachieved by subséquent addition of monodisperse magne t-icbeads coated with sheep anti-mouse IgG (Dynal, cat. no.110.02) at a cell:bead ratio of,1:1 followed by additionof anti-CD3 (clone SpvT3b) at a final dilution of 1:125000 for the experiments shown. The optimal concentration of antibody was titrated carefully in theinitial setup and parallel experiments at severaldifferent dilutions of antibody were always performed.Prolifération was analyzed by incubating cells for 72hours during which [3H]-thymidine was included for thelast 16 hours. Cells were washed and harvested ontoglass filters and subsequently analyzed by β-scintillation counting. cAMP analogs, when used, wereadded 30 min prior to.activation by addition of anti-CD3antibodies. 8-CPT-cAMP was from Sigma (St. Louis, MO).

Experimental

An on-çjoing phase II clinical trial is testing theimmunostimulatory effect of short-term treatment with anon-selective COX inhibitor (indomethacin) on surrogateparameters on T cells from HIV infected patients.According to approved protocol, patients were to receive50 mg indomethacin 3 times a day (total dose of 150mg/day) for 2 weeks with sampling at day 0, day 14 andday 28 (2 weeks after discontinuation). However, due toadverse events such as epigastrial pain and dyspepsia,and discontinuation of the study among the initialpatients, this dose had to be eut back to 25 mg 012339 - 53 - ïndomethacin 3 times a day (total dose of 75 mg/day) .Figure 11 shows T cell immune function (measured asprolifération after activation) of the 3 patients (pat. I to pat 3) that hâve so far completed the study. The 5 upper panel shows levels of prolifération after T cell

activation at start (0 days), at completion ofïndomethacin treatment (14 days) and 2 weeks thereafter(28 days) . As can be seen, patients 1 and 2 did notincrease their immune function by a non-selective COX 10 antagonist administered in vivo. However in patient 3-, T cell responsès increase approximately 2.5-fold and z persisted up to 2 weeks after discontinuation of ïndomethacin. Figure 11b, bottom panel shows T cellprolifération after incubation with a PKA-I sélective 15 cAMP antagonist, Rp-8-Br-cAMPS in vitro in cellcultures. The degree of cAMP-mediated T celldysfunction is évident from the reversai ofprolifération obtained by the antagonist (compare upperand lower panels; approx. 2-fold increase in 20 prolifération inpatients 1 and 3 at ail time points ' whereas no effect in patient 2) . It is clear from Fig. II that Ïndomethacin did not hâve a convincing effect,which may be attributed to the lack of COX-2 selectivityas well as to dose-limitations due to adverse events. 25 EXâMELE-1 HIV patients show marginal effects after. administration of non-selective Cox inhibitor jn vivo (continuation ofthe expérimenta of Example 3) 30

Me£hQ&amp;g.

The methods used were as described in Example 3.

Experimental 35 Results from 7 patients in an on-going phase II clinicaltrial (continuation of Example 3) that receivedïndomethacin 25 mg three times a day perorally for 14 □12339 - 54 - days in addition to triple combination therapy is shownin Figure 12. Patients 1-3 correspond to thosedescribed in Example 3. The problem with administrationof indomethacin is adverse events as described above(Example 3) that limit the dose to 25 mg three times aday. At this permissive dose, the effects of this non-selective COX inhibitor are marginal. After 14 days oftreatment only two of seven patients had clearlyelevated T cell immune function measured asprolifération after T cell activation whereas onepatient had decreased immune function and four patientshad minor changes. Two weeks after^discontinuâtion ofindomethacin, five of seven patients had elevated immuneresponsiveness compared to day 0. However, only twopatients had a more than two-fold increase in T cellprolifération. EXAMPLE 5

Cox-2 inhibitors improve immune function of MAIDS T cells in vitro

Methods

The methods used in the prolifération assay were asdescribed in Example 1. The PGE2 assay was as describedin Example 1.

Experimentais

Prolifération Assay

Mixed lymph node cells were isolated from MAIDS mice 17weeks post-infection. Cells were activated by anti-CD3antibodies to induce prolifération of T cells, and [3H] -thymidine incorporation was examined after 72 hours as ameasure of immune function. Prolifération of cells fromMAIDS-infected mice was again only 5 to 20% of the Tcell prolifération of uninfected cells (2000 to 12000cpm in MAIDS cells vs. mean of 55000 cpm in cells from 012339 - 55 - uninfected mice) . However, when rofecoxib (Figure 13)or celecoxib (Figure 14) were added to the cultures thisincreased the prolifération of cells from MAIDS-infectedmice two- to three-fold in a concentrâtion-dependentmanner. In contrast, treatraent of control cultures fromuninfected mice with rofecoxib or celecoxib did notincrease prolifération (0.8- to 1.0-fold increase in thepresence of COX-2 inhibitors, i.e. no increase, notshown). In T cells from MAIDS mice, the concentrationof rofecoxib and celecoxib that produced a half-maximaleffect (ED5O) was approximately 0.01 μΜ for rofecoxiband 0.03 μΜ for celecoxib. The fact that sub-raicromolarconcentrations are effective, clearly indicate that theobserved increase in immune response is mediated viainhibition of COX-2, and not COX-1 which is inhibitedonly at micromolar concentrations of rofecoxib andcelecoxib (values from Warner et al., 1999, PNAS USA, 96, p7563-7568). Thus, reversai of inhibited T cellimmune function by rofecoxib and celecoxib resuits indecreased PGE2 production in the mixed cultures andthereby lowered T cell cAMP levels via inhibition ofCOX-2. PGE2 production

The effect of the COX-2 inhibitors rofecoxib andcelecoxib on PGE2 levels was also analysed. As can beseen from Figure 15, crude lymph node cells from MAIDSmice secreted 5 to 6-fold more PGE2 than lymph node cellsfrom healthy mice (see also Fig 6) . Furthermore, PGE2levels in response to LPS increased 8-10 fo.ld ininfected compared to approximately 2-fold in uninfectedmice. When cells were incubated in the presence of COX-2 inhibitors rofecoxib or celecoxib, the PGE2 sécrétionof MAIDS lymph node cells was similar to that ofuninfected cells. The effect of indomethacin (compareprolifération in Fig. 7) is included as control. 012339 - 56 - EXAMPLE 6

Cp.x.-Z......inhibi-tar improves Immune function of maids t . cells in vivo 5 ftfetApcte .and Experimental

Infected mice (17 weeks post-infection) were treated forone week per os (i.e. orally) with a dose of rofecoxibcorresponding to the recommended dose for use in humans(and taking into account the 7-fold higher clearance in 10 rodents) . MAIDS mice normally develop an immunoproliferation syndrome with enlarged lymph nodesand spleen. In accordance with this, untreated infectedanimais had an average spleen weight of 1.3 g and anaverage weight of pooled lymph nodes of 1.7 g. In 15 contrast MAIDS mice receiving rofecoxib for 7 days hadaverage spleen weights of 0.8 g and average weight ofpooled lymph nodes of 0.3 g, indicating reversai oflymphoprolifération. 2 0 The results are shown in Figure 16. When T cell immune function was assessed in crude lymph node cells frominfected treated and untreated mice, it was clear thatwhereas untreated infected animais had anti-CD3 inducedprolifération in the range of 2000 to 10000 cpm (average 25 7300 cpm) , infected mice that received rofecoxib for one

week had T cell responses to anti-CD3 that wereincreased 2.7- to 5.6-fold compared to infected,untreated mice. Furthermore, whereas infected,untreated mi-ce demonstrated increased anti-CD3 induced T 30 cell prolifération in the presence of Rp-8-Br-cAMPS,this 2- to 3-fold effect was lost in the mice treatedwith rofecoxib, indicating that the treatment withrofecoxib in vivo lowered PGE2 levels and reversed cAMP-mediated inhibition of T cell function. 1 h 012339 - 57 - EXAMPLE 7

In. vivo treatment of MAIDS mice with rofecoxib or C-elecoxib increasea T-cell responses,, to. anti-CD3 and immune responses . Experimental

Infected mice were treated with rofecoxib and celecoxibcorresponding to the recommended dose for use in humans(and taking into account the 7-fold higher clearance inrodents, 3 and 20 mg/kg/day, respectively) . Parentéraladministration' was accomplished by intraperitoneallyinjzecting Cox-2 inhibitore formulated in intralipid.

The results are shown in Figure 17.

When T cell immune funetion was assessed in crude lymphnode cells from infected treated and untreated miceafter 18 to 20 days of infection, it was clear thatwhereas untreated infected animais had anti-CD3 inducedprolifération in the range of 10000 cpm, infected micethat received rofecoxib for 18 to 20 days had T cellresponses to anti-CD3 that were increased approximatelytwo-fold compared to infected, untreated mice.

Similarly, celecoxib improved immune responses in cellsfrom the majority of the group of mice injected toapproximately 3-fold over untreated, uninfeeted mice. EXAMPLE 8

In vivo treatment of MAIDS mice with. me.loxicam increases T-cell immune function

Methods and Experimental

Infected and healthy mice were treated with 2.8mg/kg/day meloxicam, which corresponds to therecommended dose for use in humans when taking intoaccount the 7-fold higher clearance in rodents.Parentéral administration was accomplished by 1 012339 - 58 - subcutaneous implantation of osmotic pumps filled withwater-soluble meloxicam injection compound. T cellfunction was assessed and the résulté are shown inFigure 18.

When T cell immune function was assessed in crude lymphnode cells from treated and control (PBS)-treatedinfected mice after 2 weeks of treatment, it was clearthat whereas PBS-treated, infected animais had anti-CD3induced prolifération in the range of 500 cpm, infectedmice that received meloxicam for 14 days hetd T cellimmune responses to anti-CD3 that were significantlyincreased compared to infected mice that received onlyPBS (Fig. 18a, more than 10-fold; p<0.05).

When meloxicam was added back to the cell culturesduring the 3-day in vitro T cell prolifération assay toprevent release from the in vivo inhibition by meloxicamand thereby réactivation of COX-2, the immune responsein the meloxicam-treated group was two-fold higher thanwithout addition of meloxicam in vitro (p=0.005) andcompared to that of MAIDS mice that received PBS in vivothe effect was again significant (Fig. 18b, p<0.05).

In contrast, only MAIDS mice that received PBS in vivoand not meloxicam-treated mice demonstrated increasedimmune responses when the PKA type ï-sélective cAMPantagonist, Rp-8-Br-cAMPS, was added to the anti-CD3stimulated mixed lymph node cultures in vitro (Fig. 18c) . The fact that the effect of cAMP antagonist isabsent in meloxicam-treated MAIDS mice indicates that invivo meloxicam treatment reduces or removes thecAMP-induced immunodeficiency of MAIDS and restoresimmune function.

Claims (25)

1. 59 012339 .GlftAmst

   1. Use of a COX-2 inhibitor which inhibits theenzymatic activity of COX-2 or dérivative or 5 pharmaceutically acceptable sait thereof in the préparation of a médicament for treating or preventingHIV or a related virus or AIDS.
2. 2. The use as claimed in daim 1 wherein said t 10 médicament is for administratiori to humans ou conç>anionor agri cultural animais, l f
3. 3. The use as dàimed in daim l or 2 wherein saidCOX-2 inhibitor has. a COX-1:COX-2 selectivity ratio of 15 >5, preferably >50, according to the WHMA assay at ICW.
4. 4 . The use as claitned in any one of daims 1 to 3wherein said COX-2 inhibitor ±s a methansulphonamideefcher, a methansulphonamide thioether or a diaryl 20 heterocycle,
5. 5. The use as claimed in any one of daims 1 to 4wherein said COX-2 inhibitor is a compound of generalformula A:

   wherein X représente an oxygen or sulphur atom or alkyl group,preferably a -CH2- group; Rj représente a cycloalkyl or aryl group which mayoptionally be substituted by one or more groups or 35 012339 - σο - atoms, -preferably by one or more halogen atoms; and Ra, R3, R4 and Rs independently represent a hydrogen atom,a nitro or acyl group or an alkyl group which mayoptionally be substituted‘by one or more groupa or atomsor altera^tively R2 and Ra, R3 and R, or R< and R5 togetherwith the intervening carbon atoms form a cyclopentanonegroup; or a dérivative or phàrmaceutically acceptable saittbereof. f
6. 6. The use as claimed in claim 5 wherein X is anorygen atom.
7. 7. The use as claimed in claim 5 or 6 wherein Ra is anaryl group or an aryl group substituted with one or morefluorine atome,. or a cycloalkyl group.
8. 8. The use as claimed in any one of daims 5 to 7wherein. Rz and R3 are hydrogen atoms and R4 is an -N02 or-COCH3 group.
9. 9. The use as claimed- in any one qf daims 5 to 8wherein R2 is a hydrogen àtom and R3 and R« together forma cyclopentanone group.
10. 10. The-use as claimed in any one of daims 5 to 9wherein said COX-2 inhibitor is flosulide, NS-3 98·,nimesulide, FK 3311, CGP 28232 or L-745 337.
11. 11. The use as claimed in any one of .claima 1 to 4wherein said COX-2 inhibitor is a compound of general .formula B: 012339

    wherein Y represent b a cyclic group, preferably a oxazolyl,isojcazolyl, thienyl, dihydrofuxyl, furyl, pyrrolyl, 10 pyrazolyl, thiazolyl, imidazolyl, isothiazolyl.,cyclopentenyl, phenyl or pyridyl group; ~ n îb an integer f rom 0 to 3; 15 m is an integer from û to 4; Rs représenta a ketocyclyl, cycloalkyl or aryl group,which. group may optionally be substitut ed by one or moregroupa or atome, preferably by one or more halogen 20 atoms; R, each independently represent a substituent wbidh maybe any functional group, preferably a hydrogen orhalogen atom, or an alkyl group, which alkyl group may 25 be substituted by one or raore groupa or atoms; Rg représente an 'alkyl group or nhr10; Rs représenta a halogen atom; and Rio représenta a hydrogen atom or an alkyl groupoptionally substituted by one or more groups or atoms,preferably by an acyl group; or a dérivative or a pharmaceutically acceptable saitthereof. 35 012339 - 62 -
12. 12. The use as clàimed in daim 11 wherein Re is -ΝΗ2 or-CH3.
13. 13. The use as daimed in claim 11 or 12 wherein Y is a5 pyrazolyl, furyl or thienyl group.
14. 14. The uBe as clàimed in any one of daims 11 to 13wherein Re is an aryl group optionally eubetituted. withone or more fluorine atoms. 10 ~ „
15. 15. The use as claimedin any one of daims 11 to 14wherein n is 1 or 2.

    10. The use as daimed in any one of daims 11 to‘1515 wherein R, is a bromine atom, an acyl group or a substituted alkyl group.
16. 17. -The use. as daimed in any one of daims 11 to 16wherein eaid Cox-2 inhibitor is celecoxib, rofecoxib, 20 DuP-637, SC-58125, DFP, DFU or MF tricyclic.
17. 18. The use as.daimed in any one of daims 1 to 4wherein said COX-2 inhibitor is· a non steroidal anti-inflammatory drug (NSAID) dérivative. 25
18. 19. The use as daimed in any one of daims 1 to 18wherein said COXr-2 inhibitor is diisopropylfluorophosphate, L-745337, rofecoxib, NS 39B, SC 58125, etodolac, meloxicam, celecoxib or nimesulide. 30
19. 20. The use as daimed in claim 19 wherein said COX-2inhibitor is rofecoxib.
20. 21. The use as daimed in claim 19 wherein said COX-2 35 . inhibitor is celecoxib. 012339 .- 63 -
21. 22. The use as claimed in. claim 19 wherein said COX-2•inhibitor is meloxicam.
22. 23. A pharmaceutical composition comprising a COX-2 5 inhibifcor which inhibits fcha anzymatic activity of COX-2or dexivative or pharmaceutically acceptable saitthereof as defined in any one of daims 1 to 22 and apharmaceutically acceptable diluent, carrier orexcipient for use as a médicament, preferably for 10 fcreating or ' preventing a disorder as defined in daims 1 or 2. · ~ ' ’ * .24- A pharmaceutical composition as defined in claim 23 .additioaally conprising one or more additional COX-2 15 inhibitors, dérivatives or pharmaceutically acceptablesalts thereof and/or one or more additional activeingrédients.
23. 25. A product comprising a COX-2 inhibitor which 20 inhibits the enzymatic activity of COX-2 or dérivativeor pharmaceutically acceptable sait thereof as definedin any one of daims 1 to 22 and one or more additionalCOX-2 inhibitorsz dérivatives or pharmaceuticallyacceptable salts thereof and/or one or more additional 25 active ingrédients as a combined préparation for siraultaneous, saparate or séquentiel use in fcreating orpreventing a disorder as defined in claim 1 or 2.
24. 26. Use of a pharmaceutical composition as defined in 30 'claim 23 or 24 in the préparation of a médicament for treating or preventing a disorder' as defined in daims 1or 2.
25. 27. Use of a COX-2 inhibitor which inhibits the enzymatic activity of COX-2 or 35 . dérivative or pharmaceutically acceptable sait thereof as defined in any one of daims 1 to 22 or a pharmaceutical composition as defined in daims 23 or 24 in the manufacture of amédicament for treating or preventing a disorder as defined in claim 1 or 2. î·,